P306: PAX5 DEFICIENCY AND GERMLINE SUSCEPTIBILITY TO PEDIATRIC LEUKEMIA
Authors: F. Auer, A. Escudero, M. Sipola, A. Viitasalo, M. Morcos, U. A. Friedrich, A. Pandyra, A. Borkhardt, M. Takagi, J. Hauer
Journal: HemaSphere (2022)

Background: Somatic mutations affecting the PAX5 gene are common in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, germline PAX5 mutations are extremely rare. While it is known that PAX5 acts as an indispensable master regulator of proliferation and differentiation in B-cells, the pre-leukemic setting mediated by reduced PAX5 levels is still poorly understood. Aims: To investigate the genetic and immunological effect of inherited PAX5 germline mutations in humans and to characterize the effect of Pax5 heterozygosity on B-cell development utilizing single-cell RNA Sequencing in mice. Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Sequencing, as well as whole exome Sequencing technology. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 and B-ALL development, including one family with a novel amino acid substitution p.Gly183Arg. Exome analysis of both families together with one family harboring PAX5 p.Gly183Ser previously reported by Auer et al., (Leukemia, 2014) yielded no additional common germline variants that could explain the incomplete penetrance in the observed leukemia development. A study of the respective B-cell differentiation comparing PBMCs from unrelated non-carriers, healthy carriers, and patients revealed a significant decrease of intermittent B-cells and memory B-cells in carriers and patients compared to non-carriers (Student’s T-test; p<0.05 and p<0.01, respectively). Finally, immunological studies performed using bone marrow samples from a patient and an unrelated control showed a reduction in the number of mature B cells, suggesting a block of the B-cell differentiation at the pre-B stage in the patient. In line, a comprehensive flow cytometry analysis of the B-cell development in the bone marrow of healthy Pax5 heterozygous (Pax5+/-) mice, showed a robust and age independent enrichment of the pre-BII population (B220+CD19+IgM-CD25+) compared to their Wildtype (WT) littermates (Student’s t-test; p=0.003). Bulk RNA-Sequencing of sorted preB-II cells (3 WT vs. 3 Pax5+/- mice) revealed deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs. Subsequent scRNA-Sequencing (4 WT vs. 4 Pax5+/- mice, pool of 20,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) confirmed a delayed BCR assembly and showed a skewing of light chain rearrangements towards the lambda isotype in Pax5+/- mice. Summary/Conclusion: Overall, our data provide evidence for B-cell dysregulations mediated by reduced Pax5 levels in the germline of mice and men. Ultimately these characterizations will help to advance the understanding of molecular mechanisms and susceptible populations associated with B-ALL. In turn, these findings are important to address the far more frequent somatic PAX5 mutations with the goal to prevent or treat a significant proportion of childhood leukemias.